The present invention relates to plant genetic engineering and particularly to plastid transformation in higher plants. The invention provides a novel promoter sequences useful for the expression of foreign genes of interest in various plant species.
Chloroplast genes are transcribed by an RNA polymerase containing plastid-encoded subunits homologous to the xcex1, xcex2 and xcex2xe2x80x2 subunits of E. coli RNA polymerase. The promoters utilized by this enzyme are similar to E. coil "sgr"70-promoters, consisting of xe2x88x9235 and xe2x88x9210 consensus elements (G. L. Igloi and H. Kossel, Crit. Rev. Plant Sci. 10, 525, 1992; W. Gruissem and J. C. Tonkyn, Crit. Rev. Plant. Sci. 12:-19, 1993) Promoter selection by the plastid-encoded RNA polymerase is dependent on nuclear-encoded sigma-like factors ((Link et al. 1994, Plant promoters and transcription factors, Springer Verlag, Heidelberg, pp 63-83). In addition, transcription activity from some promoters is modulated by nuclear-encoded transcription factors interacting with elements upstream of the core promoter (L. A. Allison and P. Maliga, EMBO J., 14:3721-3730; R. Iratni, L. Baeza, A. Andreeva, R. Mache, S. Lerbs-Mache, Genes Dev. 8, 2928, 1994). These factors mediate nuclear control of plastid gene expression in response to developmental and environmental cues.
There has been speculation of the existence of a second transcription system in plastids. However, direct evidence to support such a speculation has heretofore been unavailable. Identification of a novel second transcription system in plastids represents a significant advance in the art of plant genetic engineering. Such a system enables greater flexibility and range in plant species available for plastid transformation, and facilitates tissue specific expression of foreign proteins and RNAs via constructs that can be manipulated by recombinant DNA techniques.
This invention provides DNA constructs and methods for stably transforming plastids of multicellular plants. The DNA constructs of the invention extend the range of plant species that may be transformed and facilitate tissue specific expression of foreign genes of interest.
According to one aspect of the invention, DNA constructs are provided that contain novel promoter sequences recognized by a nuclear encoded plastid (NEP) RNA polymerase. The DNA construct contains a transforming DNA, which comprises a targeting segment, at least one cloning site adapted for insertion of at least one foreign gene of interest, the expression of the foreign gene of interest being regulated by a promoter recognized by a NEP polymerase, and a plastid selectable marker gene.
The use of promoter elements recognized by plastid encoded plastid (PEP) RNA polymerase for enhancing expression of foreign genes of interest is another aspect of the instant invention. Like the constructs described above, these constructs also contain a targeting segment, and a cloning site for expression of a foreign gene of interest.
The promoters recognized by plastid encoded plastid RNA polymerase have been well characterized in photosynthetic tissues such as leaf. In contrast, the nuclear-encoded polymerase transcription system of the present invention directs expression of plastid genes also in roots, seeds and meristematic tissue. In most plants, including maize, cotton and wheat, plant regeneration is accomplished through somatic embryogenesis (i.e., involving meristematic tissue). In a preferred embodiment of the invention, efficient plastid transformation in these crops will be greatly facilitated, through the use of the NEP plastid transcription system, promoters and polymerases of the present invention.
The NEP promoters of the invention are incorporated into currently available plastid transformation vectors and protocols for use thereof, such as those described in U.S. Pat. No. 5,451,513 and pending U.S. application Ser. No. 08/189,256, and also described by Svab and Maliga., Proc. Natl. Acad. Sci. USA, 90, 913 (1993), the disclosures of which are all incorporated herein by reference. To obtain transgenic plants, plastids of non-photosynthetic tissues are transformed with selectable marker genes expressed from NEP promoters and transcribed by the nuclear-encoded polymerase. Likewise, to express proteins of interest, expression cassettes are constructed for high level expression in non-photosynthetic tissue, using the NEP promoter transcribed from the nuclear-encoded polymerase. In another aspect of the invention, PEP promoters of the invention are incorporated into currently available plastid transformation vectors and protocols for use thereof.
In yet another aspect of the invention, the NEP transcription system also may be combined with the "sgr"70-type system through the use of dual NEP/PEP promoters.